title: Multisteroid LC-MS/MS assay for glucocorticoids and androgens, and its application in Addison's disease.
abstract: Liquid chromatography-mass spectrometry (LC-MS/MS) offers superior analytical specificity compared to immunoassays, but is not available in many regions and hospital due to expensive instrumentation and tedious sample preparation. Thus, we developed an automated, high-throughput LC-MS/MS assay for simultaneous quantification of 10 endogenous and synthetic steroids targeting diseases of the hypothalamo-pituitary-adrenal-axis and gonads.
  
  
  
  Deuterated internal standards were added to 85µl serum and processed by liquid-liquid-extraction. Cortisol, cortisone, prednisolone, prednisone, 11-deoxycortisol, dexamethasone, testosterone, androstenedione and progesterone were resolved by ultra-high pressure chromatography on a reversed-phase column in 6.1 minutes, and detected by triple-quadrupole MS. The method was used to assess steroid profiles in women with Addison's disease (AD, n=156) and blood donors (BD, n=102).
  
  
  
  Precisions ranged 4.5-10.1% RSD, accuracies 95-108%, and extraction recoveries 60-84%. The method was practically free of matrix effects and robust to individual differences in serum composition. Most postmenopausal AD women had extremely low androstenedione below 0.14 nmol/L and median testosterone 0.15 nmol/L [interquartile range 0.00-0.41], considerably lower than postmenopausal BD (1.28 nmol/L [0.96-1.64] and 0.65 nmol/L [0.56-1.10], respectively). AD women in fertile years had androstenedione 1.18 nmol/L [0.71-1.76] and testosterone 0.44 nmol/L [0.22-0.63], approximately half of levels found in BD of corresponding age.
  
  
  
  This LC-MS/MS assay provides highly sensitive and specific assessments of glucocorticoids and androgens with low sample volumes, and is suitable for endocrine laboratories and research. Its utility was demonstrated in a large cohort of women with AD, and the data suggest that women with AD are particularly androgen deficient after menopause.
authors.affiliation: P Methlie, Department of Clinical Science, University of Bergen, Bergen, Norway.
authors.forename: Paal
authors.initials: P
authors.lastname: Methlie
authors.forename: Steinar Simon
authors.initials: SS
authors.lastname: Hustad
authors.forename: Ralf
authors.initials: R
authors.lastname: Kellmann
authors.forename: Bjørg
authors.initials: B
authors.lastname: Almås
authors.forename: Martina Moter
authors.initials: MM
authors.lastname: Erichsen
authors.forename: Eystein
authors.initials: E
authors.lastname: Husebye
authors.forename: Kristian
authors.initials: K
authors.lastname: Løvås